Study of the genes TACI and ICOS in patients with B lymphoproliferative disorders

Purpose: TACI and BAFFR might participate in the survival of CLL cells, protecting them from apoptosis through NF-κB. Mutations of TNFRSF13B, TNFRSF13C and ICOS genes have been associated with antibody deficiencies. The aim of this study was to detect the mutations of TNFRSF13B gene in Greek patients with antibody deficiencies and to determine the expression of TNFRSF13B/TACI, TNFRSF13C/BAFFR and ICOS on CLL cells and their contribution to the phenotype and the prognosis of the disease. Materials and methods: Samples from 53 patients with antibody deficiencies, 94 patients with CLL, 30 patients with NHL and 315 healthy individuals, were collected. The mRNA and protein levels of TNFRSF13B and TNFRSF13C were estimated by qRT-PCR and flow cytometry, respectively. BAFF and APRIL serum levels were estimated by ELISA. In CLL, the prognostic factors CD38 and the hypermutation status of IGHV gene were determined using flow cytometry and PCR-sequencing, respectively. CLL cells were stimulated by BAFF and APRIL and the apoptosis status was estimated by flow cytometry using an Annexin V-FITC/7-AAD (7-AAD) kit. The molecular analysis of TNFRSF13B gene was performed using PCR-sequencing and the detection of the polymorphisms TNFRSF13B-P251L, TNFRSF13B-V220A, TNFRSF13C-P21R and TNFRSF13C-H159Y using PCR-RFLP. Results: Μutations of TNFRSF13B gene are found both in patients and healthy individuals. CLL patients displayed signicantly lower expression of TACI (Median: 7,4%, IQR:2,1-20,8) compared to NHL patients (Median: 31,5, IQR: 9,6-61,8), p<0,001 and lower MFI of BAFFR compared to healthy controls (Median: 6,1 IQR:4,6-7,5 vs Median: 9,8 IQR:6,7-14,5 respectively). Serum BAFF was absent in the majority of CLL patients. Additionally, CLL cells with low or absent TACI expression were more susceptible to apoptosis in vitro after stimulation with all mitogens. TNFRSF13B/TACI expression and TNFRSF13C/BAFFR expression were not signicantly associated with the presence of autoimmunity, the infection risk and the prognosis of CLL. Conclusions: The mutations of TNFRSF13B are not causative factors for the apperance of antibody deficiencies. Τhe low TACI expression suggest that CLL may derive from CD5+ B cell populations rather than memory B cells and low BAFFR MFI could be used for the detection of minimal residual disease of CLL. These findings should be taken into account in the case of anti-TACI and anti-BAFFR therapeutic approaches.Εισαγωγή: Οι υποδοχείς TNFRSF13B/TACI και TNFRSF13C/BAFFR ενεργοποιούν τον NF-κΒ στα νεοπλασματικά Β-κύτταρα προστατεύοντας τα πιθανόν από την απόπτωση. Μεταλλάξεις των γονιδίων TNFRSF13B, TNFRSF13C και ICOS, έχουν ενοχοποιηθεί στην παθογένεση των αντισωματικών ανεπαρκειών και στην εμφάνιση λεμφοϋπερπλασιών. Σκοπός της διατριβής ήταν η μελέτη του TNFRSF13B σε ανοσοανεπαρκούντες ασθενείς και των TACI, BAFFR και ICOS σε ασθενείς με ΧΛΛ. Υλικό και Μέθοδοι: Συλλέχθηκαν δείγματα 53 ασθενών με αντισωματικές ανεπάρκειες, 94 με ΧΛΛ, 30 με NHL και 315 φυσιολογικών μαρτύρων. Η έκφραση TACI και BAFFR σε πρωτεϊνικό επίπεδο ελέγχθηκε με κυτταρομετρία ροής ενώ σε μεταγραφικό επίπεδο με qRT-PCR. Στους ασθενείς με ΧΛΛ μελετήθηκαν προγνωστικοί δείκτες της νόσου, όπως η έκφραση CD38 με κυτταρομετρία ροής καθώς και οι υπερμεταλλάξεις της βαριάς αλυσίδας των ανοσοσφαιρινών με PCR-sequencing. Στον ορό ασθενών με ΧΛΛ και φυσιολογικών μαρτύρων προσδιορίστηκαν τα BAFF και APRIL. Η λειτουργικότητα του TACI ελέγχθηκε μετά από καλλιέργεια των ΧΛΛ κυττάρων παρουσία BAFF και APRIL. Στους ασθενείς με αντισωματικές ανεπάρκειες έγινε PCR-sequencing του γονιδίου TNFRSF13B, ενώ στα δείγματα ασθενών με ΧΛΛ και φυσιολογικών μαρτύρων έγινε ανίχνευση των πολυμορφισμών TNFRSF13B- P251L, TNFRSF13B- V220A, TNFRSF13C- P21R και TNFRSF13C- Η159Υ. Αποτελέσματα: Γενετικές βλάβες του TNFSRF13B εμφανίζονται σε ανοσοανεπαρκούντες και σε φυσιολογικά άτομα. Τα Β-κύτταρα ΧΛΛ ασθενών εξέφραζαν BAFFR, αλλά με σημαντικά χαμηλότερη ένταση φθορισμού συγκριτικά με τα φυσιολογικά Β-κύτταρα (Median:6,1 IQR:4,6-7,5 vs Median:9,8 IQR:6,7-14,5), p<0,001. Η πλειονότητα των ασθενών με ΧΛΛ παρουσίαζε χαμηλή έκφραση TACI (Median:7,4%, IQR:2,1-20,8), σε σχέση με τους ασθενείς με NHL (Median:31,5, IQR: 9,6-61,8), p<0,001. Στον ορό των ασθενών τα επίπεδα BAFF ήταν χαμηλά και τα επίπεδα APRIL αυξημένα συγκριτικά με τους φυσιολογικούς μάρτυρες. Δε διαπιστώθηκε συσχέτιση της έκφρασης TACI και BAFFR με την παρουσία αυτοάνοσων εκδηλώσεων, λοιμώξεων ή την πρόγνωση της ΧΛΛ.Συμπεράσματα: Οι μεταλλάξεις του γονιδίου TNFSRF13B δεν είναι αιτιολογικές για την εμφάνιση αντισωματικών ανεπαρκειών. Η χαμηλή έκφραση TACI ισχυροποιεί την υπόθεση προέλευσης του λευχαιμικού κυττάρου από τον Β1-υποπληθυσμό. Το πρότυπο έκφρασης του BAFFR από τα νεοπλασματικά Β-κύτταρα, το καθιστά χρήσιμο δείκτη για τον έλεγχο της υπολειπόμενης νόσου. Τα αποτελέσματα μπορούν να ληφθούν υπόψιν στην περίπτωση αντι-TACI και αντι-BAFFR θεραπευτικών προσεγγίσεων

Transient ischemic attacks as the first presentation of JAK2-V617F positive chronic myeloproliferative neoplasm

Several studies have shown that thrombotic events may underlie a latent or subclinical myeloproliferative neoplasm (MPN) and precede its definite diagnosis by 1–2 years. An early diagnosis of patients with MPN, especially those with thrombotic events in the latent MPN phase, would be beneficial for their management, preventing further morbidity and improving their quality of life. For the majority of these cases, the location of thrombosis is mainly in the splanchnic major veins, while ischemic stroke and cerebral venous thrombosis are rarely observed. In this report, we present a female patient with transient ischemic attacks who suffered from a latent MPN, on the basis of a positive testing for the JAK2-V617F mutation

Detection of activated STAT5 (p-STAT5), DPF3 staining and mRNA levels in CLL.

<p><b>(A) Flow cytometry analysis:</b>Indicative plots of peripheral blood cells analysis are shown (healthy individual with low <i>DPF3</i> expression levels (3.6) in upper panels and CLL patient with high <i>DPF3</i> expression levels (19.69) in lower panels). Dot plots on the left indicate percentages of gated cells used on the analysis (A: lymphocytes in pink, B: monocytes in green and C: granulocytes in red). Histograms on the right indicate p-STAT5 levels in lymphocytes (pink), monocytes (green) and granulocytes (red). Isotype control (IgG1) is depicted in grey. <b>(B) Levels of p-STAT5 in the peripheral blood cell subpopulations analyzed by flow cytometry:</b> The boxes represent the interquartile range containing 50% of values in lymphocytes, monocytes and granulocytes of healthy individuals (n = 4) and CLL patients (n = 10). The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers. A line across the box indicates the median value for each group. The statistical significance of difference is noted on the charts. <b>(C) </b><b><i>DPF3</i></b><b> expression levels (mRNA) in granulocytes and monocytes: </b><i>DPF3</i> expression levels were measured in granulocytes and monocytes of CLL patients (n = 5) and healthy individuals (n = 4) by real time PCR. Bars demonstrate mean and standard error of the mean (SEM) values of expression. The statistical significance of the differences in expression is indicated with an asterisk (*0.01≤P<0.05). <b>(D) Immunofluorescence detection of nuclear p-STAT5:</b> Granulocytes with low to high intensity nuclear p-STAT5 staining were scored as positive for the nucleus. Granulocytes with absent nuclear staining and low to high intensity cytoplasmic staining were considered positive for the cytoplasm (***P<0.001). <b>(E) Immunofluorescence detection of nuclear DPF3:</b> Scoring of nuclear DPF3 staining (detected as puncta in the nucleus) was performed on granulocytes positive for p-STAT5 staining in the cytoplasm and/or the nucleus. Granulocytes with low to high intensity puncta in the nucleus were considered positive (***P<0.001). <b>(F) Immunofluorescence detection of both nuclear p-STAT5 and DPF3:</b> For co-localization of DPF3 (red) and p-STAT5 (green) nuclear staining, granulocytes with green and red, and/or yellow staining puncta (representing p-STAT5 and DPF3 co-localization) were scored (*P = 0.05). In D, E and F: Scoring was performed in granulocytes showing p-STAT5 staining in the nucleus, the cytoplasm or both; bars demonstrate mean and SD values obtained at least in three different slides or regions of the same slide; the statistical significance of the difference between samples with low (healthy individuals) and high (CLL patients) <i>DPF3</i> expression is shown; <i>DPF3</i> expression levels (mRNA) are shown below the samples; ND: not determined.</p

STAT5 binding to human <i>DPF3</i> promoter.

<p><b>(A) STAT5a binding to human <i>DPF3</i> promoter in granulocytes:</b> Cross-linked chromatin from granulocytes of a CLL patient and a healthy individual was used in ChIPs with anti-STAT5a antibody. <b>(B) STAT5a binding to human </b><b><i>DPF3</i></b><b> promoter in CLL cell lines:</b> Cross-linked chromatin from EHEB and JVM-2 cells was used in ChIPs with anti-STAT5a antibody. In A and B, IgG was utilized in parallel with anti-STAT5a antibody, as control. Two sets of primers were used: One set specific for the amplification of the <i>DPF3</i> promoter containing a STAT5 motif and one set of negative control primers (neg) for amplification of a region lacking STAT5 motifs. Bars represent mean and SD values of specific enrichments (fold differences) versus input obtained at least in three independent experiments. The statistical significance of enrichment for promoter sequences versus the negative control region (<b>*</b>0.01≤P<0.03) and for patient versus healthy individual (<b>**</b>P = 0.007) is indicated; a-STAT5a: anti-STAT5a.</p

Expression of <i>DPF3</i> gene in patients with hematologic malignancies.

<p>Means of <i>DPF3</i> expression levels (relative mRNA levels), standard errors (SE) and range for each patient cohort and healthy controls are shown. ?Statistical significance refers to comparison with the expression levels in the PB (P₁) or BM (P₂) of healthy controls (Mann-Whitney U test). Statistically significant P values are depicted in bold. Considering that in AML, ALL and CML, a rather similar infiltration of PB and BM by neoplastic cells is observed, a comparison with both groups of healthy controls (PB and BM) was performed.</p

The novel STAT5 target gene <i>Dpf3:</i> Expression levels (in wild type and knock-down cells) and STAT5a, STAT5b binding.

<p><b>(A) Expression levels of <i>Dpf3</i>:</b> Expression levels (mRNA) were measured by real time PCR in Ba/F3 cells deprived of IL-3 for 6 h (starved) and stimulated with IL-3 for 30 min and 3 h. The deprived of IL-3 cells were set as 1. Bars demonstrate mean and standard deviation (SD) values obtained at least in three independent experiments. The statistical significance of the difference in expression between 30 min stimulated and deprived of IL-3 cells is indicated with asterisks (**P = 0.005). <b>(B)</b><b>STAT5a and STAT5b binding to </b><b><i>Dpf3</i></b><b>:</b> Cross-linked chromatin from Ba/F3 cells deprived of IL-3 for 6 h and stimulated with IL-3 for 30 min was used in ChIPs with anti-STAT5a or anti-STAT5b antibody. Three sets of primers were used for each gene. One set specific for the amplification of the isolated genomic region/cloned sequence (seq) containing at least one TTCN_3/4GAA motif, one set specific for the promoter of each gene (pr) containing at least one TTCN₃GAA motif and one set of negative control primers (neg) for amplification of a region lacking TTCN_3/4GAA motifs. IgG was utilized in parallel with anti-STAT5a or anti-STAT5b antibodies, as control. Bars demonstrate mean and SD values of specific enrichments (fold differences) versus input obtained at least in three independent experiments. The statistical significance of enrichment versus the negative control region is indicated with asterisks (ns: not significant, *P = 0.01, **P = 0.002, ***P<0.001). <b>(C)</b><b>Expression levels of </b><b><i>Dpf3</i></b><b> in cells with knock-down of STAT5a, STAT5b or both:</b> Expression levels (mRNA) of <i>Dpf3</i> were measured in Ba/F3 cells with knock-down of STAT5a, STAT5b or both. The cells were deprived of IL-3 for 6 h (starved) and stimulated with IL-3 for 30 min. Expression levels were measured by real time PCR and compared with Ba/F3 cells in the deprived of IL-3 state, transduced with scrambled shRNA and set as 1. Bars demonstrate mean and SD values obtained at least in three independent experiments. The statistical significance of the difference in expression between knock-downs and the respective scrambled control is indicated with asterisks (ns: not significant, *P = 0.02, **P = 0.005, ***P<0.001).</p

Methodologies and genome distribution of the sequences.

<p><b>(A) Scheme of the Methodologies followed:</b> Streptavidin chromatin precipitations and ChIPs followed by streptavidin precipitations were performed in Ba/F3 cells expressing BirA or BirA/bioSTAT5a (left). Single and double ChIPs with anti-STAT5a antibody were performed in Ba/F3 cells (right). All cells were deprived of IL-3 for 6 h and subsequently stimulated with IL-3 for 30 min. <b>(B) Genome distribution of the sequences:</b> Genome distribution of the sequences from the ChIP followed by streptavidin precipitation library is shown on the left and from the double ChIP library on the right. The pie charts show the genome distribution of the sequences relative to the nearest neighbor gene, located within <3 kb and 3–5 kb relative to TSS (transcription start site), introns, exons and intergenic regions.</p

Identification of a STAT5 Target Gene, Dpf3, Provides Novel Insights in Chronic Lymphocytic Leukemia

STAT5 controls essential cellular functions and is encoded by two genes, Stat5a and Stat5b. To provide insight to the mechanisms linking hematologic malignancy to STAT5 activation/regulation of target genes, we identified STAT5 target genes and focused on Dpf3 gene, which encodes for an epigenetic factor. Dpf3 expression was induced upon IL-3 stimulation in Ba/F3 cells, while strong binding of both STAT5a and STAT5b was detected in its promoter. Reduced expression of Dpf3 was detected in Ba/F3 cells with Stat5a and Stat5b knock-down, suggesting that this gene is positively regulated by STAT5, upon IL-3 stimulation. Furthermore, this gene was significantly up-regulated in CLL patients, where DPF3 gene/protein up-regulation and strong STAT5 binding to the DPF3 promoter, correlated with increased STAT5 activation, mainly in non-malignant myeloid cells (granulocytes). Our findings provide insights in the STAT5 dependent transcriptional regulation of Dpf3, and demonstrate for the first time increased STAT5 activation in granulocytes of CLL patients. Novel routes of investigation are opened to facilitate the understanding of the role of STAT5 activation in the communication between non-malignant myeloid and malignant B-cells, and the functions of STAT5 target genes networks in CLL biology